Duquesnoy, RJ

Eiras, G

Makowka, L

Starzl, TE

Todo, S

Zeevi, A

English

D-Scholarship@Pitt

INHIBITORY EFFECTS OF FKS06 IN COMBINATION WITH CsA ON HUMAN LYMPHOCYTE ALLOPROLIFERATIVE RESPONSES 1 ,2 G. Eiras*, A. Zeevi*, S. Todo*·, L. Makowka**, T.E. Starzl*· and R.J. Duquesnoy* From the Departments of Pathology* and Surgery*. University of Pittsburgh, PA fOOTNOTES 1. Supported by NIH grants AI23467 and HL36416 2. Address correspondence to: Adriana Zeevi, Ph.D. University of Pittsburgh Division of Clinical Immunopathology Room 5711, One Children's Place Pittsburgh, PA 15213-3417 ---, FK506 (Fi<) is a novel lmmunosuppressl ve arug lsolatea from Streptomyces tsukubaensis. Its 1n vitro effect on lymphocyte proliferation is several hundred fold greater than that of Cyclosporine A (CsA). Studies ..... ere conducted on the inhibitory effect of combinations of FK and CsA on lymphocyte proliferation in vitro. The secondary proliferative response (PLT) of alloreactive T cells ..... as significantly inhibited by a combination of 0.06 nglml of FK and 1 nglml CsA. At these low concentrations, these drugs were ineffective. The synergistic e f fee t bet wee n F K and C sAw a s a Iso de 0, 0 n s t rat edt h r 0 ugh sequential exposure of alloreactive lymphocytes to Fi< followed by CsA. Pretreatment of IL-2 expanded alloreactive cells with Fi< yielded cell cultures whose PLT activity ..... as considerably more sensitive to low doses of CsA than control cells or CsA pretreated cells. These findings provide evidence for a synergism between the inununosuppressive effects of Fi< and CsA. The combination of low dose FK plus CsA inununosuppression may benefit organ transplant outcome. Recent studies have described a new, potent immunosuppressive drug FK 506 which induces considerable prolongation of allograft survival (1,2,3). Fi< is isolated from Streptomyces tsukubaensis in Osaka, Japan. Its molecular structure resembles the macrolide family with a molecular weight of 822 daltons (4). The inununosuppressive effect of Fi< can be demonstrated in primary mixed lymphocyte reaction (MLR), and in secondary proliferation of al10reactive T cells generated from MLR or propagated from organ transplant biopsies (5,6). Studies on lymphocyte proliferat10n 1n prlmary MLR have previously shown that inhibition was achieved with doses of Fl< that were several hundred fold lower than those of CsA (5,6). An example of the difference between Fl< and CsA is illustrated in Table 1. Significant inhibition (>70\) of the alloreactive response, measu red in pr imed 1 ymphocy te tes t ( PLT), of ali ver biopsy propagated lymphocyte culture was achieved with a low dose of 0.25 ng/ml Fl<. On the contrary, a 200 fold higher concentration of CsA was necessary to obtain the same level of inhibition. In vivo studies have shown a similarly potent effect of Fl< on the prolongation of transplant survival in experimental animals (7-9). Polypharmaceutical therapy is currently widely used in organ transplantation to avoid side effects associated with high doses of a single agent. Studies have been conducted to determine in vitro synergism between Fl< and CsA. Our first approach was to add to lymphocyte cultures low doses of these drug combinations. As shown in Figure 1, the combination of 0.06 nglml Fl< and 10 ng/m1 of CsA induced a significant inhibition (40\) of the PLT reactivity of liver biopsy grown cells. On the other hand neither drug was effective at these low doses. In addition, a 10 fold lower dose of CsA (1 ng/ml) 0.06 ng/ml of fK induced a significant PLT inhibition indicating that fK acts synergistically with a relatively wide dose range of CsA (Figure 1). In contrast, a two-fold reduction of the fK dose (to 0.03 ng/m1) added to 10 ng/ml of CsA greatly diminished the synergistic inhibitory effect between these drugs (10,11). · ~~ ... ~-... --K------~KI- .. ~K-.. -.-----------.. ---- ... ~--K~K The second approach to study the synergistic effect between these two drugs was through sequential exposure of alloreactive lymphocytes to FK followed by CsA. Because FK and CsA have no effect on IL2 induced proliferation of activated T cells (10), experiments were set up whereby heart transplant biopsy grown lymphocytes were expanded in IL2 in the presence of various concentrations of FK (0.1 uglml and 0.01 ug/ml) or CsA (10 ug/ml) . After four days 1n culture, these pretreated cells showed similar PLT reactivity and IL-2 responsiveness as untreated control cultures. On the other hand, FK pret reated cultures showed increased CsA sensitivity when tested 1n PLT (Table 2). For instance, the PLT response of alloreactive T cells pretreated with 0.1 nglml FK was inhibited by 78\ with a low dose of 0.05 nglml of CsA. On the other hand, pretreatment with 10 nglml of CsA did not effect the CsA sensitivity of a subsequent PLT (Table 2). These findings of a synergism between FK and CsA have an important clinical application in that low doses of these drug combinations may have less adverse side effect but are still effective in increasing transplant survival. Indeed, Todo and his colleagues, using the experimental models of heterotopic heart transplantation in rats and renal allografts in dogs, have recently demonstrated that the most efficient immunosuppressive protocols are combinations of low doses of FK with subtherapeutical doses of CsA and prednisone (7). The mechanism of the synergism between FK and CsA is unknown. Preliminary observations at our institution have shown, that FK enhances CsA uptake by peripheral blood lymphocytes (12). It is possible that the synergism between FK and CsA might be related to lymphocyte membrane binding of these drugs. -. : K~-r K~ .1 .1. Ochiai T, Nakajima K, Kagale H, Nagata H, Suzuki T, Asano T, Vematso T, Goto T4 Hori S, Kenmochi T, Nakagoori T, Isono K: 2 • 3. Effect of a new immunosuppressive agent, FK506 on heterotopic cardiac allotransplantation in the rat. Transp1. Proc. 19:1284-1286, 1987. Lee P, Murase N, Todo S, Makowka L, Starzl T: The immunosuppressive effects of FR 900506 in rats heterotopic cardiac allografts. Surgical Communications 1:325, 1987. receiving Research Todo S, Hakowka L, Starzl T: Immunosuppression wi th FR 900506 for cani ne kidney and liver t r ansplan ts. Research Communications 1:330, 1987. Surgical 4. Goto T, Kino T, Hatanaka H, Nishiyama M, Okuhara M, Kohsaka M, Aoki Hand Imanaka H: Discovery of FK506, a novel immunosuppressant isolated' from Streptomyces Tsukubaensis. Transpl. Peoc. 19 suppl 6, 1987, pp. 4. 5. Kino T, Inamura N, Sakai F, Nakahara K, Goto T, Ohuhara M, Kohsaka M, Aoki Hand Ochiai T: Effect of FK506 on human mixed lymphocyte reactions in vitro. s u pp 1 6, 1987, pp. 36. Transp1. Proc. 19 6. Zeevi A, Duquesnoy R, Eiras G, Todo S, Makowka Land Starzl 7 • TE: In vitro immunosuppressive effects of FR900506 on human T lymphocyte activation. Todo S, Demetrius AJ, Casavi1la A, Cemaj S, Surge Res. Corrunun. 1:315, 1987. Veda Y, Imventarza 0, Okuda K, Ghalab A, Mazzaferro V, Rhoe BS, Tonghua Y, Makowka Land Starzl TE: Canine kidney transplantation with FKS06 alone or in combination with --.-- .------------.. -----cyclosporine and steroids. 1987, pp. 57. Transpl. Peoc. 19 Suppl 6, 8. Ochiai T, Nagata M, Nakajima K, Sakamoto K, Asano T, Isonok. Prolongation of canine renal allograft survival by treatment with fK506. Transpl. Proc. 19, Supp1.6, 1987, pp.53. 9. Todo S, Podesta L, Chapchap P, Kahn 0, Pan CE, Veda Y, Okuda K, fmventa~za 0, Casavilla A, Demetris AJ, Makowka L, Starzl TE: Orthotopic liver transplantation in dogs receiving fK506. Transpl. Proc. :9, Supple 6, 1987, pp. 64. 10. Zeevi A, Duquesnoy R, Eiras G, Rabinowich H, Todo S, Makowka Land Starzl TE: Immunosuppressive effect of FKS06 on in vitro lymphocyte activation: synergism with cyslosporine A. Transpl. Proc. 19 suppl 6, 1987, pp. 40. 11. Zeevi A, Duquesnoy R, Eiras G, Rabinowich H, Todo S, Makowka Land Starzl TE. In vitro immunosuppressive effects of fKS06 in combination with other drugs. Transpl. Proe. In press, 1987. 12. Sanghvi A, Warty V, Zeevi A, Diven W, Duquesnoy R, Makowka L and Starzl TE: fKS06 enhances cyclosporine uptake by peripheral blood lymphocytes. Transpl. Proc. 19 suppl 6, 1987, pp. 45. 13. Zeevi A, Fung JJ, Zerbe T, Kaufman C, Rabin S, Griffith B, Hardesty Rand Duquesnoy RJ: Allospeeificity of activated T cells grown from endomyocardial biopsies from heart transplant patients. Transplantation 41:620, 1986. FIGURE 1 Synergism between the immunosuppressive effects of FK506 and CsA. The donor-specific PLT reactivity of· liver biopsy propagated lymphocyte culture was tested in the presence of combinations of low doses of fK (0.06 ng/ml) and CsA (10 ng/ml or 1 ng/ml). 

Inhibitory effects of FK506 in combination with CsA on human lymphocyte alloproliferative responses

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Inhibitory effects of FK506 in combination with CsA on human lymphocyte alloproliferative responses

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